Dissecting PKA activation of mTORC1 and its function in adipose tissue

剖析 mTORC1 的 PKA 激活及其在脂肪组织中的功能

• 批准号: 10091138
• 负责人: SHEILA COLLINS
• 金额: $ 6.52万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-22 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10091138
• 关键词: Adipocytes; Adipose tissue; Alanine; Amino Acids; Antibodies; Area; Aspartate; Behavioral; Biochemical; Biogenesis; Biological; Body fat; Body mass index; Brown Fat; CRISPR/Cas technology; Calories; Catecholamines; Cell Culture Techniques; Cell model; Cells; Chronic Disease; Clinical; Clothing; Complex; Country; Cyclic AMP; Cyclic AMP Receptor Protein; Cyclic AMP-Dependent Protein Kinases; Data; Data Set; Desire for food; Development; Disease; Endocrine System Diseases; Energy Metabolism; Engineering; Epidemic; Event; FDA approved; FRAP1 gene; Fatty Acids; Fatty acid glycerol esters; Follow-Up Studies; Generations; Genes; Genetic; Genetic Transcription; Glucose; Goals; Growth Factor; Human; In Vitro; Insulin; Insulin Resistance; Intervention; Investigation; Knock-in; Knowledge; Laboratory Animals; Life Style; Lipolysis; Longevity; Mechanics; Metabolic; Metabolic Diseases; Metabolic syndrome; Metabolism; Mitochondria; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Overweight; PPAR gamma; Pathway interactions; Peripheral; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Population; Process; Production; Proteins; Proteomics; Protons; Public Health; Raptors; Receptor Activation; Regulation; Resistance; Respiration; Ribosomal Protein S6 Kinase; Risk; Rodent; Role; Route; Safety; Scanning; Signal Pathway; Signal Transduction; Sirolimus; Site; Source; Stable Isotope Labeling; Sympathetic Nervous System; Techniques; Technology; Testing; Therapeutic; Thermogenesis; Tissues; Weight Gain; Work; absorption; beta-adrenergic receptor; cell type; comorbidity; comparative; cost; experimental study; fat burning; fighting; gain of function; gain of function mutation; health economics; improved; in vivo; inhibitor/antagonist; insight; insulin sensitivity; kinase inhibitor; loss of function mutation; novel; novel strategies; obesity treatment; p38 Mitogen Activated Protein Kinase; phosphoproteomics; prevent; programs; reconstitution; transdifferentiation; uncoupling protein 1

Project Summary: Obesity is at epidemic proportions in the US. Over 60% of the population is either overweight (Body Mass Index [BMI] ≥25 to <30 kg/m2) or obese (BMI ≥30 kg/m2), placing them at risk for a large number of chronic diseases, including insulin resistance, metabolic syndrome, and type 2 diabetes. The annual costs of obesity exceed $100 billion, making it one of the most significant public health and economic issues facing the country. Unfortunately, the treatment of obesity is unsatisfactory. Lifestyle and behavioral approaches have a modest, and often transient, effect while FDA-approved therapeutic options targeting appetite or fat absorption have poor tolerability and, in some cases, safety concerns. Thus, there is a critical need for novel approaches to treat obesity. Agents acting via peripheral mechanisms to increase energy expenditure would be valuable. The sympathetic nervous system (SNS) is well-known as an activator of brown adipose tissue (BAT) and the “browning” of cells in white adipose tissue (WAT) depots to increase uncoupled mitochondrial respiration and energy expenditure. Our earlier work established a signaling cascade from β-adrenergic receptors (βARs)  cAMP  protein kinase A (PKA)  p38 MAP kinase (MAPK) to drive the transcription of brown adipocyte genes such as uncoupling protein-1 (UCP1), PPAR-gamma coativator-1α (PGC-1α), and the broader program of mitochondrial biogenesis. We have discovered that the mTOR complex-1 (mTORC1) components mTOR and Raptor are phosphorylated by PKA. This is a highly novel observation, since the `canonical' pathway to mTORC1 is through growth factors and insulin. From in vivo studies in mice we find that blockade of mTORC1 with rapamycin, or genetic deletion of Raptor specifically in adipose tissue, suppresses the ability of the βAR pathway in increase the amount of UCP1-expressing `beige' adipocytes within white fat depots, and dampens UCP1 expression and respiration in interscapular brown fat. The ability of laboratory animals and humans to expand these `beige' adipocytes is closely correlated with resistance to weight gain and improved insulin sensitivity. We have identified the phosphorylation sites on mTOR and Raptor and propose to test the physiological consequences of cells and mice in which Ser791 of Raptor is changed to either Alanine (loss-of-function mutation) or Aspartate (gain-of-function) by engineering a `knock-in' genetic construct. Since we now show that there are two distinct routes to activation of mTORC1, we are also taking an unbiased proteomic approach to identify the unique phosphorylation substrates of mTOR resulting from PKA activation vs insulin. Altogether, these experiments will shed important mechanistic and physiological insight into the steps needed for `beige' cell expansion, as well as the broader ability of the PKA mTORC1 pathway to function in other cell types.

项目总结： 在美国，肥胖症的流行程度很高。超过60%的人口要么超重(身体质量 体重指数[BMI]≥25至30 kg/m2)或肥胖(BMI≥30 kg/m2)，使他们面临患上大量慢性 疾病，包括胰岛素抵抗、代谢综合征和2型糖尿病。肥胖症的年度成本 超过1000亿美元，使其成为该国面临的最重要的公共卫生和经济问题之一。 不幸的是，肥胖症的治疗并不令人满意。生活方式和行为方式有适度的， 通常是暂时的，而FDA批准的针对食欲或脂肪吸收的治疗方案有 耐受性差，在某些情况下，安全问题。因此，迫切需要新的方法来 治疗肥胖症。通过外围机制来增加能量消耗的代理将是有价值的。这个 交感神经系统(SNS)是众所周知的棕色脂肪组织(BAT)的激活剂，而 白色脂肪组织(WAT)储存库中细胞的“褐变”，以增加未偶联的线粒体呼吸和 能源支出。我们早期的工作建立了β-肾上腺素能受体(β-AR)的信号级联 CAMP-蛋白激酶A--p38-MAPK对棕色脂肪细胞转录的影响 解偶联蛋白-1(UCP1)、PPAR-γ衣壳蛋白-1α(PGC-1α)等基因，以及更广泛的程序 线粒体的生物发生。 我们发现mTOR复合体-1(MTORC1)成分mTOR和Raptor被磷酸化 由PKA提供。这是一个非常新奇的观察结果，因为通向mTORC1的典型途径是通过生长。 因子和胰岛素。在小鼠体内的研究中，我们发现用雷帕霉素或基因阻断mTORC1 脂肪组织中特异的Raptor基因的缺失，抑制了βAR途径增加 白色脂肪库中表达UCP1的“米色”脂肪细胞的数量，并抑制UCP1的表达和 肩胛骨间棕色脂肪的呼吸。实验动物和人类扩大这些“米色”的能力 脂肪细胞与抵抗体重增加和改善胰岛素敏感性密切相关。 我们已经确定了mTOR和Raptor上的磷酸化位点，并建议测试生理上的 在细胞和小鼠中，Raptor的Ser791改变为丙氨酸(功能丧失)的后果 突变)或天冬氨酸(功能获得)通过设计“Knock-in”基因结构。既然我们现在展示的是 有两种不同的途径可以激活mTORC1，我们也采取了一种公正的蛋白质组学方法来 确定由PKA激活和胰岛素引起的mTOR的独特的磷酸化底物。总而言之， 这些实验将为“米色”所需的步骤提供重要的机械学和生理学见解。 细胞扩增，以及PKAmTORC1通路在其他细胞类型中发挥作用的更广泛的能力。